中文版 | English
题名

基于 CRISPR 的无扩增核酸检测系统及其在 感染性疾病可视化诊断上的应用

其他题名
UNAMPLIFIED NUCLEIC ACID DETECTION SYSTEM BASED ON CRISPR AND ITS APPLICATION IN VISUAL DIAGNOSIS OF INFECTIOUS DISEASES
姓名
姓名拼音
NIU Dan
学号
12132014
学位类型
硕士
学位专业
0856 材料与化工
学科门类/专业学位类别
0856 材料与化工
导师
张博
导师单位
生物医学工程系
论文答辩日期
2023-05-17
论文提交日期
2023-07-03
学位授予单位
南方科技大学
学位授予地点
深圳
摘要
当突发性、传染性疾病大流行时,快速确定感染源并与其他病原体进行区分,迅速建立大规模筛查体系,切断传播链是应对疫情的首要任务。当前主流的基于扩增的核酸检测技术灵敏度高,但扩增依赖昂贵的检测设备,难以应用于临床快检。因此开发一种兼具高灵敏度、高通量的检测性能和便携、快速的实用性能的免扩增核酸检测体系,有望为感染性疾病的现场快速检测提供新的思路。
CRISPRClustered Regularly Interspaced Short Palindromic Repeats)家族中的Cas12a 蛋白在识别到靶标 DNA 时会被激活非特异性内切酶活性,会随机切断其周围的单链 DNAssDNA),结合分子信标探针能够实现信号放大。通过将 CRISPRCas12a 体系与酶联免疫信号放大体系结合,本项目构建了一种二次信号放大的免扩增核酸检测策略。首先 ssDNA 末端标记辣根过氧化酶(HRP),并将其固载于纳米磁珠表面。将 Cas12a 蛋白与 gRNAguide RNA)孵育形成核糖核蛋白复合体(RNP),当体系中存在靶标 DNA 时,gRNA 能够识别靶标 DNA,以此激活 Cas12a 蛋白,非特异性地切割体系中的 ssDNA。体系中由 HRP 标记、连接在磁珠表面的ssDNA 被激活的 Cas12a 蛋白降解,其末端标记的 HRP 会被释放到溶液中,随后利用磁分离移除纳米磁珠,上清中的游离 HRP 能够催化 3,3’5,5’-四甲基联苯胺 (TMB)显色实现高灵敏度(100 fM)的检测。 为了实现不依赖仪器的 Point-of-CarePOC)检测,我们进一步构建具有大斯托克斯位移性质的过氧化物荧光探针。过氧化物荧光探针被 HRP 与过氧化物催化后,能够在紫外光激发条件下,发射可见光波段荧光。结合 Cas12a 蛋白与酶联免疫信号放大体系,大斯托克斯位移的过氧化物荧光探针可实现肉眼可见的无扩增核酸检测。本课题通过将荧光分子设计与 CRISPR-Cas 技术结合,拟摆脱当前核酸检测技术对连续变温和实时荧光信号采集装置的依赖,实现无扩增的病原体核酸检测,有望满足感染性疾病对现场快速检测的临床需求。
关键词
语种
中文
培养类别
独立培养
入学年份
2021
学位授予年份
2023-06
参考文献列表

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牛丹. 基于 CRISPR 的无扩增核酸检测系统及其在 感染性疾病可视化诊断上的应用[D]. 深圳. 南方科技大学,2023.
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